| Vitamin B12 (cobalamin, Cbl) is an essential nutrient in humans and functions as the cofactor for methylmalonyl-CoA mutase (Mut) and methionine synthase (MS). Genetic disorders linked to intracellular processing of cobalamin to methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), the cofactor forms required for MS and Mut, respectively, have been defined through complementation analysis. Currently, all eight complementation groups, designated cblA-G and mut, have had their corresponding genes identified.;Secondly, I investigated the well documented but enigmatic differential response of cblC fibroblasts and patients to OHCbl compared to CNCbl. For this investigation I used recombinant cblC (MMACHC) protein, including wild-type protein as well as forms with the G147D early-onset cobalamin unresponsive and R161Q late-onset cobalamin responsive cblC mutations. I demonstrated that wild-type MMACHC was able to bind OHCbl and CNCbl with the same tight affinity (Kd = 5.7 μM), that MMACHC-G147D did not bind either form and that MMACHC-R161Q bound OHCbl with wild-type affinity but was deficient in its ability to bind and decyanate CNCbl. Additionally, by mutating MMACHC-H122, the histidine suggested to be integral to the B12-binding motif, I found that binding to OHCbl and CNCbl was reduced but not ablated, suggesting this histidine is not absolutely required for binding. These data pointed to the inability to bind cobalamin as the basis for functional deficiency in early-onset (G147D) cblC and differential binding of OHCbl and CNCbl as the basis for the better response to OHCbl with late-onset (R161Q) cblC. However, since MMACHC-R161Q was able to bind OHCbl with wild-type affinity, the nature of the defect in cblC patients with this allele remained unclear. To explore this further, I investigated the thermostability of MMACHC-R161Q in comparison to wild-type MMACHC. I found that MMACHC-wt and MMACHC-R161Q were both very thermolabile proteins, with melting temperatures (Tm) of 39.3 ± 1.0°C and 37.1 ± 0.7°C, respectively. Additionally, I surveyed the effect on stability upon binding to different forms of cobalamin and found that, at 10 μM cofactor concentration, both MMACHC-wt and MMACHC-R161Q were stabilized in the order AdoCbl > MeCbl > HOCbl > CNCbl. Significantly, as for HOCbl, MMACHC-R161Q was stabilized less than MMACHC-wt after binding each of these forms of cobalamin. Together, these results suggested that MMACHC is an unstable protein that is normally stabilized in the cell by Cbl binding and the R161Q mutation doubly destabilizes this protein by decreasing its stability in the apo- form and decreasing the stability gain it achieves when bound to cobalamin.;Finally, I investigated the possible interaction between MMAA, a cobalamin processing protein of unknown function, and Mut, the enzyme responsible for AdoCbl utilization, using E. coli as well as human cells. Using recombinant human MMAA and Mut, I found that while MMAA retained the ability to bind GTP and GDP and confirmed that our recombinant Mut binds AdoCbl, these proteins did not form a complex in vitro as judged by native gel electrophoresis or size-exclusion chromatography under any conditions tested, including the addition of non-hydrolyzable GTP. However, following over-expression and pull-down of MMAA, Mut could be detected by Western blot analysis, indicating that these proteins do interact in vivo. These results suggested that human Mut and MMAA interact in vivo but the conditions for this interaction are not easily duplicated in vitro. (Abstract shortened by UMI.).;I first investigated the intracellular localization of MSR, a protein known to be involved in the cytosolic reduction of cobalamin but suggested to also function in the mitochondrion. Evidence of a possible mitochondrial function consisted of a putative mitochondrial leader on an alternate splice isoform of MSR as well as in vitro evidence that MSR could physically interact with MMAB, a mitochondrial protein, to produce AdoCbl. I demonstrated that, while there indeed was a second splice isoform of MSR, this isoform lacked a functional mitochondrial leader sequence. |
